[The cell wall of Bacillus brevis producing gramicidin S, a cyclodecapeptide antibiotic]. / Issledovanie obolochki Bacillus brevis--produtsenta tsiklodekapeptidnogo antibiotika gramitsidina S.

Gramicidin S is tritiated in Bacillus brevis G.-B. intact cells by activated tritium atoms (which is indicative of its surface localisation). The cell wall protein is tritiated very weakly, which shows that it is screened, apparently, by gramicidin adsorbed on its surface. The cell wall protein is not a glycoprotein and its interaction with exogenous gramicidin S causes cell aggregation. As follows from the Rf value after the chromatographic separation of B. brevis lipids, the reaction of staining, and the data of H-NMR spectroscopy for the fraction of phospholipids, the main membrane phospholipid is an anionic acetylated phosphatidyl ethanolamine.

[Action of benzohydrothiochromylium salts on bacterial membranes]. / Deistvie solei benzogidrotiokhromiliia na membrannyi apparat bakterii.

Cultivation of Staphylococcus 209-P and Micrococcus lysodeikticus cells on media containing new antibacterial preparations--iodide and trifluoroacetate derivatives of benzohydrothiochromylium resulted in a remarkable lesion of the membrane respiratory apparatus, i.e. the amounts of membrane polypeptides, the specific concentration of cytochromes, the activities of reductases and oxidases--NADH, malate and lactate decreased. Profound changes in the cell cytology were observed.

[Composition and properties of the outer cell wall layer in Bacillus brevis--the producer of gramicidin S]. / Sostav i svoistva naruzhnogo sloia kletochnoi stenki Bacillus brevis--produtsenta gramitsidina S.

Two membrane antigens were found by cross immunoelectrophoresis in the cell walls of Bacillus brevis var. G.-B., R form, which started to synthesize gramicidin S (20 mg per 1 ml of cultural broth). The cell wall contained no membrane components in cells at the beginning of the logarithmic growth phase. The protein with a molecular mass of 100 kDa is a component of the cell wall outer layer. The protein is not digested by trypsin or pronase when it comprises the cell walls of cells synthesizing gramicidin S. In the preparation of isolated cell walls, this protein becomes susceptible to the action of the above proteases only when the peptidoglycan layer is broken down by lysozyme. Electron microscopy of cells treated with proteases and shadowed with a metal revealed that many cells lacked the cytoplasm. Therefore, the outer layer of B. brevis R cell wall contains small regions susceptible to the action of protease along with regions composed of the 100 kDa protein and resistant to these enzymes. It is possible that the small regions contain membrane components.

[Organization and chemical composition of the cell wall of gramicidin S-producing Bacillus brevis]. / Osobennosti organizatsii i khimicheskogo sostava kletochnoi stenki Bacillus brevis - produtsenta gramitsidina S.

The morphology of cells and cell walls was studied in the Bacillus brevis G.-B. R form during its growth and gramicidin S accumulation in it. The membrane apparatus became more complicated and certain other morphological changes were detected in the cells with aging. The cell wall was rather complex even in young cells and consisted of three electron-dense layers where the external and internal layers had an ordered structure. Only the external layer underwent some modifications in the course of growth and these coincided in time with the beginning of intensive gramicidine S biosynthesis. However, the three-layer structure of the cell wall and the ordered organization of the external and internal layers remained unchanged. A preparation of cell walls and preparations of their external and internal layers were isolated from cells synthesizing gramicidine S in the amount of 20 micrograms/ml of the cultural broth. An acid protein having the molecular mass of 100 kD was shown to be the major component of the external layer according to the data of electrophoresis in PAAG with SDS. The middle layer was sensitive to lysozyme, did not have a ordered structure on electron micrographs, and consisted mainly of peptidoglycan.

[The effect of nitrofurans on the membrane apparatus of coccal bacteria]. / Deistvie nitrofuranov na membrannyi apparat bakterii kokkovoi gruppy.

Secondary membrane effects on the membrane apparatus of coccus bacteria were being studied. Cultivation of Micrococcus lysodeikticus and Staphylococcus aureus cells on subbacteriostatic concentrations of nitrofurans results in a lower biosynthesis of many membrane proteins, as well as in inhibiting the activity of respiratory enzymes, i. e. the specific concentration of cytochromes and specific activity of NADH-, malate-, lactate oxidases and some reductases drop. Some cytological changes were revealed, when cells were grown on solafur, furazolidone, and furacriline.

[Structuro-functional properties of the bacteria Bacillus brevis in relation to the accumulation of gramicidin S in cells]. / Izuchenie strukturno-funktsional'nykh osobennostei bakterii Bacillus brevis v sviazi s nakopleniem v kletakh gramitsidina S.

The culture of Bacillus brevis var. G-B R-form was grown in the presence of beta-phenyl-beta-alanine, the inhibitor of gramicidin S synthesis, is characterized by enhanced endogenous respiration and the DPI-reductase activity as compared to the culture synthezising antibiotic. The increased synthesis of the antibiotic in the region of the culture transition from the logarithmic growth phase to the linear one is associated with a decrease in the number of viable cells despite the fact that the culture on the whole does not die but continues to grow. The membranes prepared from young gramicidin S-free cells and from the cells enriched with the antibiotic possess identical electron micrograph images, IR spectra and protein sets as determined by polyacrylamide gel electrophoresis in a Na-DS system. However, in young cell membranes NADH and succinate dehydrogenase are insensitive to gramicidin S and only malate dehydrogenase is inhibited by this antibiotic. In aged cell membranes the activities of all mentioned dehydrogenases are suppressed. Malate dehydrogenase from young cells is weakly inhibited by thyrotrycin obtained from Bac. brevis ATCC 10068; succinate dehydrogenase is entirely insensitive to this antibiotic, while NADH-dehydrogenase is almost completely inhibited by it. The specificity of action on the respiratory chain of peptide antibiotics synthesized by the cells of one strain of Bac. brevis is suggestive of a possible regulatory role of these peptides in the metabolism of the producent. Hence the accumulation of gramicidin S which is adsorbed on the membrane and destroys the respiratory chain function to the cause of the low rate of oxygen uptake by the culture of Bac. brevis var. G-B R-form and of the low activities of DPI-reductases.

[Existence of boundary lipids in reconstituted hydrophobic protein-lipid system]. / Sushchestvovanie pogranichnykh lipidov v rekonstruirovannow sisteme gidrofobnyi belok--lipid.

A fraction of hydrophobic proteins (9, 14, 18 kD) soluble in a chloroform-methanol mixture (2:1) has been isolated from Micrococcus lysodeikticus bacterial membranes. The proteins obtained were introduced into proteoliposomes at a protein/lipid weight ratio ranging from 0.1 to 0.25 in combination with the fluorescent probe pyrene or the spin probe 2-(14-carboxytetradecyl)-2-ethyl-4.4-dimethyl-3-oxasolidinyloxyl. The excimertization of pyrene upon direct excitation of its molecules (gamma excit.=338nm) and under conditions of energy transfer from the excited protein chromophores to pyrene (gamma excit.=286nm) and the spin-spin exchange between the spin probe molecules was investigated. The experimental results suggest that the hydrophobic protein molecules are surrounded by a structurally heterogenous lipid area containing up to 3.3 mg of lipid per l mg of protein. The maximal expression of structural heterogeneity was observed at the minimal content of protein in the proteoliposomes. Treatment with the membranotropic antibiotic gramicidin S resulted in disappearance of lateral heterogeneity of lipids in the constituted system and in lipid aggregation in bacterial membranes. It is assumed that the aggregability of membrane proteins depends on the structural rearrangement of some part of lipid bilayer around them.

[Proteolysis as an approach to the study of protein distribution in the membrane of Micrococcus lysodeikticus]. / Proteoliz kak podkhod k izucheniia raspolozheniia belkov v membrane Micrococcus lysodeikticus.

Treatment of M. lysodeikticus protoplasts with subtilisin or pronase did not affect their permeability and led to a digestion of 20--30% of protein. DS-Na electrophoresis of protoplast membranes resulted in disappearance of three protein bands. This suggests that the outer surface of M. lysodeikticus protoplasts contains three proteins other than respiratory chain enzymes, which are subjected to an attack by proteinases. Treatment of the M. lysodeikticus membranes, isolated by osmotic shock, with proteinases resulted in a digestion of 20--50% of protein. The factors preventing the interaction between the membrane components (e.g. decrease of Mg2+ concentration, ultrasound, KCl, EDTA and particularly detergents) favoured the proteolysis; however, the bulk of the proteins remained insensitive to the effect of proteinases. The membranes pretreated with DS-Na or chlorophorm--methanol mixture proved to be good substrates for proteinases. Treatment of the membrane fraction with proteolytic enzymes allowed to obtain some data on localization of respiratory chain enzymes in the membrane stroma of M. lysodeikticus. Thus, cytochrome c is localized nearer to the membrane surface than cytochromes a and b, while malate dehydrogenase is plunged deeper into the membrane stroma as compared to NADH dehydrogenase.

[Lateral heterogeneity of bacterial membranes]. / Lateral'naia neodnorodnost' bakterialnoi membrany.

Isolated membranes of M. lysodeikticus were rapidly frozen and disrupted in a Hughes press. After disruption the fragments were centrifuged at 144000 g for 1 hour and part of the supernatant just above the pellet was subjected to isopycnic centrifugation in a continuous sucrose density gradient. It was found that the material tested was a mixture of fragments differing in their buoyant densities. These fragments also differed in their protein/lipid ratios, cytochrome content and dehydrogenase activities calculated per protein and lipid as well as in proportion of the respiratory chain enzymes. The results obtained are indicative of lateral heterogeneity of the bacterial membrane and the existence of areas in the membrane having high concentration of the respiratory chain enzymes. The latter may suggest that the system of substrate oxidation is segregated in the membrane. It is assumed that there exists in the membrane an exchange of components between different electron-transporting chains operated due to their lateral diffusion.

Manipulation of phospholipid composition of membranes with the aid of lipid exchange proteins. Incorporation of phosphatidylcholine into protoplasts of Micrococcus lysodeikticus.

Incubation of Micrococcus lysodeikticus protoplasts with phosphatidylcholine liposomes and rat liver exchange proteins (pH 5.1 supernatant fraction) resulted in replacement of about one half of the bacterial total phospholipids by phosphatidylcholine. Protoplasts modified by phosphatidylcholine showed a decreased rate of oxidation of exogenous substrates (NADH, malate) and decreased ferricyanide reductase activity as compared to the initial protoplasts. At the same time incorporation of phosphatidylcholine had no influence on the level of endogeneous respiration. Protoplasts modified by phosphatidylcholine were osmotically more stable than the initial protoplasts. After osmotic lysis of the phosphatidylcholine protoplasts their NADH (malate) oxidase and ferricyanide reductase activities were restored. Incorporation of phosphatidylcholine into membrane ghosts, obtained by osmotic rupture of the initial protoplasts had only small if any effect on the malate and NADH oxidase and dehydrogenase activities. It is concluded that phosphatidylcholine in incorporated predominantly into the outer part of cytoplasmic membrane and that proteinmediated transfer of phosphatidylcholine results in restoration of the permeability barrier due to repair of local defects in the initial protoplast membrane.

[Variation of cell membrane lipid composition by means of lipid transfer proteins. Properties of the protoplast membrane of Micrococcus lysodeikticus after incorporation of phosphatidylcholine]. / Izmenenie lipidnogo sostava kletochnykh membran s pomoshch'iu lipidperenosiashchikh belkov. Svoistva membran protoplastov Micrococcus lysodeikticus, soderzhashchikh fosfatidilkholin.

After incorporation of phosphatidylcholine (PC) into the protoplast membrane of M. lysodeikticus by protein mediated transfer from PC liposomes, the activity of some membrane bound respiratory chain enzymes was studied. It was found that incorporation of PC decreases the rates of oxidation of exogenous substrates (NADH, malate) but the level of endogenous respiration was not changed. Ferricyanidreductase activity of ghosts of M. lysodeikticus was not dependent upon the PC content of protoplasts. PC containing protoplasts showed a higher osmotic stability than unmodified protoplasts. It is concluded that the incorporation of PC into the protoplasts results in resealing, i. e. in the repair of local defects in the protoplast membrane.